Shigellae are highly human-adopted Gram-negative enterobacteria causing bacillary dysentery. The disease spreads exclusively by direct personal contact or human fecal contamination of food and water. As a result, bacillary dysentery is endemic in regions with suboptimal hygienic conditions. There are an estimated 165M cases worldwide, with as many as 1M fatalities mostly among children under the age of five Shigella was found to be the one of the most prevalent bacterial pathogen isolated in case of an acute diarrheal episode among 1-5 year-old children in Southeast Asia and sub-Saharan Africa (Kotloff et al., Bull. W.H.O. 77: 651-666, 1999). Bacillary dysentery is also common among travelers and military personnel entering endemic countries. It has long been accepted that vaccines would be crucial to control dysentery but vaccine development against Shigellae is hampered by the serotype-specific immune response, i.e upon exposure to Shigella (natural or vaccine-mediated) the immune protection is usually limited to the given serotype. The four species of the Shigella genus comprise a sum of 50 serotypes and subserotypes, which are differentiated by their LPS O-antigens.
Hong et al. (Molecular Microbiology 24:779-91, 1997) described the effect of mutations in chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance of Shigella flexneri. Mutations in the rfb and rfaL genes either eliminated the entire O-antigen side chains or produced chains of greatly reduced length.
Nagy et al. (J. Infect. Diseases 198: 1699-706, 2008) described the vaccine potential of a Salmonella enterica regulatory lipopolysaccharide mutant. Loss of the transcriptional antiterminator RfaH resulted in a heterogeneous length of LPS chains, the “gently rough” phenotype.
Regulatory protein RfaH is shown to be involved in the growth-phase dependent upregulation of long-chained (i.e. high number of O-antigen repeat) LPS molecules of S. flexneri (Carter et al. Microbiology. 2007 October; 153 (Pt 10):3499-507).
Major virulence factors of Shigellae other than LPS O-antigens are surprisingly conserved. This implies a lack of immune-mediated evolutionary pressure on these antigens substantiating the accepted view about O-antigen being the sole protective antigen. Nevertheless, there is a marked antibody response against the so-called “invasion plasmid antigens” (Ipa-s), especially following repeated exposure. These antigens encoded on the large virulence plasmid form components of a type three secretion system (T3SS) that is indispensable for invasion and hence virulence.
The structure-function analysis of the Shigella virulence factor invasion plasmid antigen B (ipaB) was disclosed by Guichon et al. (J. Bacteriol. 183:1269-76, 2001). ipaB mutants were generated to correlate function with protein subdomains.
Menard et al. (J. Bacteriol. 17518: 5899-5906, 1993) described that mutagenesis of the ipa genes ipaB, ipaC and ipaD of Shigella flexneri, resulted in the loss of invasive potential of Shigella. 
Antibodies against Ipa proteins (such as to minor conserved antigens) are considered non-protective, as otherwise cross-protection among serotypes could be triggered. Current vaccine approaches rely almost exclusively on O-antigen mediated immunity exploiting the fact that five or six serotypes would provide high protection against the majority of endemic and epidemic dysentery cases. Nevertheless, considering the fact that most of the suggested multivalent vaccines are based on either purified subunits (O-antigens) or several live attenuated bacteria with different LPS O-antigen types, they would, most probably, be too complex and hence expensive. Moreover, a partial serotype coverage is expected to induce serotype replacement due to herd immunity based immune pressure on vaccine serotypes and escape and increase in prevalence of non-vaccine serotypes, as demonstrated for other multi-serotype pathogens, such as Streptococcus pneumoniae. Therefore, an ideal Shigella vaccine is expected to provide substantial cross-protection against all circulating serotypes.
Besides Shigella, Enterotoxigenic Escherichia coli (ETEC) is a major bacterial pathogen responsible for travelers' diarrhea and represent one of the leading cause of death in children in endemic countries. Therefore efforts are undertaken to develop vaccines addressing these two pathogens simultaneously.
Travelers' diarrhea is currently treated by antibiotics; however, there is an increasing rate of resistance among Shigella strains that makes the management of the disease more and more difficult. Moreover, ETEC infections can have long-term consequences related to irritable colon syndrome. It is widely accepted that vaccination would be the most effective way to address this high unmet medical need; yet, no vaccines are currently available for the prevention of these conditions.
Two types of enterotoxins have been identified in ETEC strains, the heat labile toxin (LT) and the heat stable toxin (ST), either as ST associated with porcine disease (STp) or ST associated with human disease (STa). LT is highly homologous in structure to the cholera toxin. The A subunit is the active component of the toxin, which functions to increase the activity of adenylate cyclase. This is delivered into host cells by the B subunits, which bind to gangliosides on the cell surface. STa is a small (19 amino acid) non-immunogenic polypeptide that has guanylate cyclase stimulating activity. STm is a mutated form of ST that is non-toxic, but still immunogenic. Such STm is considered to be safely employed as vaccine antigen (Taxt et al. Infect. Immun. 78:1824-31, 2010).
It has been demonstrated that ETEC strains also produce EAST1, a heat-stable toxin similar in size and mode of action to ST but different in sequence, originally identified in enteroaggregative E. coli strains (Nataro and Kaper, Clin Microbial Rev. 11: 142-201, 1998; Zhang et al., Vet Microbial. 123: 145-152, 2007).
Zheng et al. (World J. Gastroeneterol. 11: 3411-18, 2005) constructed an asd mutant Shigella strain co-expressing CS3 and LTB/STm of enterotoxigenic E. coli. After immunization of mice by the oral route, antibodies were raised against CS3, LTB, ST, and Shigella lipopolysaccharide.
Xu et al. (Vaccine 21: 664-648, 2003) described a live attenuated invasive Shigella flexneri serotype 2a rfbF mutant as a carrier for a DNA-based HIV gag vaccine.
Noriega et al. (Infection and Immunity 67(2): 782-788, 1999) described a strategy for cross-protection against 14 Shigella flexneri serotypes, involving the use of the two serotypes 2a and 3a. The attenuated strains described are S. flexneri 2a strain CVD1207 (ΔguaB-A Δset1 Δsen) and S. flexneri 3a strain CVD 1211 (ΔguaB-A ΔvirG Δsen).
Bernardini et al. (Infection and Immunity 69(2): 1072-1083, 2001) describe mutants of Shigella flexneri 5, which are an aroC mutant and a double purE aroC mutant.
Levine et al. (Behring Institute Mitteilungen 98: 120-123, 1997) described an attenuated S. flexneri 2a strain CVD 1203 which harbors mutations in chromosomal gene aroA and plasmid gene virG, as well as an S. flexneri 2a vaccine candidate CVD 1205 that harbors a deletion mutation in guaB-A rendering it defective in nucleic acid synthesis, and a deletion mutation in virG.
It is the objective of the present invention to provide improved Shigella vaccines, in particular for the prevention of diarrheal diseases that are highly relevant for travelers to endemic countries and young children living in developing countries. Such vaccines can be based on a live attenuated Shigella flexneri vaccine strain able to heterogeneously express antigens derived from different pathogens and induce broad protection against bacterial pathogens, and particularly shigellosis.